Abstract
Glycan microarrays provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. However, the construction of current glycan microarray platforms is time consuming and expensive. Here, we report a fast and cost-effective method for the assembly of cell-based glycan arrays to probe glycan–glycan-binding protein interactions directly on the cell surface. Chinese hamster ovary cell mutants with a narrow and relatively homogeneous repertoire of glycoforms serve as the foundation platforms to develop these arrays. Using recombinant glycosyltransferases, sialic acid, fucose, and analogs thereof are installed on cell-surface glycans to form cell-based arrays displaying diverse glycan epitopes that can be probed with glycan-binding proteins by flow cytometry. Using this platform, high-affinity glycan ligands are discovered for Siglec-15—a sialic acid-binding lectin involved in osteoclast differentiation. Incubating human osteoprogenitor cells with cells displaying a high-affinity Siglec-15 ligand impairs osteoclast differentiation, demonstrating the utility of this cell-based glycan array technology.
Highlights
Glycan microarrays provide a high-throughput means of profiling the interactions of glycanbinding proteins with their ligands
The lectin-resistant Chinese hamster ovary (CHO) cell mutant Lec[2] that expresses a narrow and relatively homogenous repertoire of glycoforms is employed as the foundation platform
We demonstrate the utility of these cell-based arrays to interrogate glycanbinding proteins (GBPs) specificities and ligand tolerance directly on the cell surface
Summary
Glycan microarrays provide a high-throughput means of profiling the interactions of glycanbinding proteins with their ligands. Sialic acid, fucose, and analogs thereof are installed on cell-surface glycans to form cell-based arrays displaying diverse glycan epitopes that can be probed with glycan-binding proteins by flow cytometry. Using this platform, high-affinity glycan ligands are discovered for Siglec-15—a sialic acid-binding lectin involved in osteoclast differentiation. We demonstrate the utility of these cell-based arrays to interrogate GBP specificities and ligand tolerance directly on the cell surface This method is applied to high throughput screening for the identification of selective and high-affinity ligands of Siglecs, a family of sialic acid-binding immunoglobulin-type lectins that are differentially expressed primarily on immune cells. A high-affinity glycan ligand for Siglec-15 is discovered that can be used to modulate the differentiation of osteoclasts
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